Systems and methods for processing-nucleic acid molecules from a single cell using sequential co-partitioning and composite barcodes

Abstract

Provided herein are methods, compositions, and systems for multiplexed analysis of individual cells or cell populations. Cells encapsulated in beads and/or biomolecules are sequentially co-partitioned, allowing for analysis of two different types of biomolecules (e.g., RNA and DNA). The present invention leverages different polymer dissociation mechanisms, accompanied with barcoding of biomolecules (e.g., nucleic acid molecules) for multiplexed measurements in single cells. Sequential co-partitioning and barcode technology enables identification and quantitation of DNA and RNA from single cells.

What is claimed is: 
     
       1. A method for barcoding nucleic acid molecules of a cell, comprising:
 (a) co-partitioning a cell bead and a first bead in a first partition, wherein said cell bead is generated from said cell, wherein said cell comprises a first nucleic acid molecule and a second nucleic acid molecule, wherein said first nucleic acid molecule comprises a different nucleic acid sequence than said second nucleic acid molecule, and wherein said first bead comprises a first set of barcode molecules each comprising a first nucleic acid barcode sequence; 
 (b) subjecting said first partition to a first condition sufficient to permit (i) said first nucleic acid molecule to couple to a first barcode molecule of said first set of barcode molecules to generate a first barcoded nucleic acid molecule comprising said first nucleic acid barcode sequence, and (ii) a second barcode molecule of said first set of barcode molecules to associate with said cell bead; 
 (c) co-partitioning said cell bead associated with said second barcode molecule or derivative thereof and a second bead in a second partition, wherein said second bead comprises a second set of barcode molecules each comprising a second nucleic acid barcode sequence; and 
 (d) subjecting said second partition to a second condition sufficient to permit (i) said second nucleic acid molecule to couple to a first barcode molecule of said second set of barcode molecules to generate a second barcoded nucleic acid molecule comprising said second nucleic acid barcode sequence, and (ii) a second barcode molecule of said second set of barcode molecules to couple to said second barcode molecule to generate a composite barcode molecule comprising said first nucleic acid barcode sequence and said second nucleic acid barcode sequence. 
 
     
     
       2. The method of  claim 1 , wherein in (b) said second barcode molecule attaches to said cell bead. 
     
     
       3. The method of  claim 1 , further comprising sequencing said first barcoded nucleic acid molecule or derivative thereof, said second barcoded nucleic acid molecule or derivative thereof, and said composite barcode molecule or derivative thereof to generate sequencing reads, and using said sequencing reads to associate said first nucleic acid molecule and said second nucleic acid molecule as having originated from said cell or cell bead. 
     
     
       4. The method of  claim 1 , wherein subjecting said first partition to said first condition comprises subjecting said cell to conditions sufficient to lyse said cell. 
     
     
       5. The method of  claim 1 , wherein prior to (a), a plurality of first nucleic acid molecules is coupled to said cell bead. 
     
     
       6. The method of  claim 5 , wherein said plurality of first nucleic acid molecules is coupled to said cell bead via one or more capture nucleic acid molecules configured to capture said first nucleic acid molecule. 
     
     
       7. The method of  claim 6 , wherein said plurality of first nucleic acid molecules comprises messenger ribonucleic acid (mRNA) and said one or more capture nucleic acid molecules comprise a dT sequence. 
     
     
       8. The method of  claim 1 , wherein said cell bead comprises an interpenetrating polymer network comprising a first layer of polymer network and a second layer of polymer network. 
     
     
       9. The method of  claim 8 , wherein said first layer of polymer network dissociates by a first dissolution mechanism and said second layer of polymer network dissociates by a second dissolution mechanism, wherein said first dissolution mechanism and said second dissolution mechanism are different. 
     
     
       10. The method of  claim 9 , wherein subjecting said first partition to said first condition comprises applying said first dissolution mechanism, and wherein subjecting said second partition to said second condition comprises applying said second dissolution mechanism, wherein said first dissolution mechanism comprises a reduction or oxidation reaction. 
     
     
       11. The method of  claim 9 , wherein said first dissolution mechanism releases said first nucleic acid molecule from said cell bead without releasing said second nucleic acid molecule from said cell bead, and wherein said second dissolution mechanism releases said second nucleic acid molecule from said cell bead. 
     
     
       12. The method of  claim 8 , wherein said first layer of polymer network comprises disulfide crosslinking bonds. 
     
     
       13. The method of  claim 8 , wherein said second layer of polymer network comprises oxidizable crosslinking bonds. 
     
     
       14. The method of  claim 1 , wherein (c) comprises using a magnetic field to capture said cell bead associated with said second barcode molecule. 
     
     
       15. The method of  claim 1 , wherein said first nucleic acid molecule is selected from the group consisting of deoxyribonucleic acid and ribonucleic acid. 
     
     
       16. The method of  claim 1 , wherein said first condition and said second condition are different. 
     
     
       17. The method of  claim 16 , wherein subjecting said first partition to said first condition comprises applying a first dissolution mechanism and subjecting said second partition to said second condition comprises applying a second dissolution mechanism, wherein said first dissolution mechanism and said second dissolution mechanism are different, and wherein said first dissolution mechanism comprises a reduction or oxidation reaction. 
     
     
       18. The method of  claim 17 , wherein said first dissolution mechanism releases said first nucleic acid molecule without releasing said second nucleic acid molecule from said cell bead. 
     
     
       19. The method of  claim 1 , wherein said first partition or said second partition is a droplet. 
     
     
       20. The method of  claim 1 , wherein said first partition or said second partition is a well. 
     
     
       21. The method of  claim 1 , wherein said first bead or said second bead is a gel bead.